Mbewe et al. Parasites & Vectors (2018) 11:268 https://doi.org/10.1186/s13071-018-2840-6

RESEARCH Open Access Sticky small target: an effective sampling tool for tsetse Glossina fuscipes fuscipes Newstead, 1910 Njelembo J. Mbewe1,2*, Rajinder K. Saini1,3, Baldwyn Torto1,2, Janet Irungu1, Abdullahi A. Yusuf2 and Christian W. W. Pirk2

Abstract Background: Small targets comprising panels of blue and -treated black netting material each 0.25 × 0. 25 m have been shown to attract and kill Glossina fuscipes fuscipes Newstead, 1910 (Diptera: Glossinidae) thereby reducing its population density by over 90% in field trials. However, their attractive ability has not been fully exploited for sampling purposes. Therefore, in this study we assessed the effectiveness of using sticky small targets as sampling tools for G. f. fuscipes in western Kenya. We also determined the influence of colour on the landing response of female and male on sticky small targets. Methods: Using a series of randomised block experiments, the numbers of tsetse flies caught with sticky small targets were compared with those caught with biconical traps. A negative binomial regression was used to model fly catches. Odds ratios as measures of association between the landing response on the blue or black panel of the sticky small target and the sex of flies were obtained from a multiple logistic regression. Results: The results showed that sticky small targets caught 13.5 and 3.6 times more female and male tsetse flies than biconical traps (Z = 9.551, P < 0.0001 and Z = 5.978, P < 0.0001, respectively). Females had a 1.7 times likelihood of landing on the black panel than males (Z = 2.25, P = 0.025). Conclusion: This study suggests that sticky small targets are an effective sampling tool for G. f. fuscipes. Therefore, we recommend the use of sticky small targets as an alternative to biconical traps for observational and experimental investigations of G. f. fuscipes. Keywords: Riverine tsetse flies, Small targets, Sampling, Behaviour, Density, Surveillance

Background [3, 4]. So far, there is no vaccine and the main interven- African (HAT), also referred to tion for Gambian HAT (gHAT) is the use of case detec- as sleeping sickness is transmitted by tsetse flies har- tion and treatment programmes [4]. The method aims bouring the mature protozoan parasites, to clear the parasite in a substantial proportion of the brucei rhodesiense and T. brucei gambiense, as they take human population so that even when bitten, the a human blood meal causing Rhodesian and Gambian will not pick up any trypanosomes for further transmis- HAT, respectively [1, 2]. Tsetse flies from the palpalis sion [1]. However, infected flies can still transmit new group which occupy riverine habitats have been impli- gHAT to [4]. Further, the chronic na- cated in the transmission of over 90% of HAT cases ture of gHAT allows for silent carriers harbouring low caused by T. brucei gambiense in central and west Africa parasite levels undetectable by conventional diagnostic methods to sustain transmission foci [5–7]. Thus, inter- * Correspondence: [email protected] ventions that target the vector further reduce the risk of 1 International Centre of Physiology and Ecology, P.O Box 30772-00100, new infections from occurring [4, 8]. For con- Nairobi, Kenya 2Department of Zoology and Entomology, University of Pretoria, Private Bag trol, targets are simple, cheaper and easier to maintain X20, Hatfield, Pretoria 028, South Africa than traps [9]. Insecticide-treated targets usually greater Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mbewe et al. Parasites & Vectors (2018) 11:268 Page 2 of 7

or equal to 1.0 × 1.0 m in size are effective control tools trap which is commonly used for sampling G . f. fuscipes for the savannah tsetse fly, G. pallidipes Austen and G. and other riverine tsetse flies. We also assessed the influ- morsitans morsitans Westwood [10]. Additionally, be- ence of colour on the landing response of male and fe- havioural studies that aimed at developing cost effective male G. f. fuscipes on the sticky small target. targets for the main vectors of gHAT, G. fuscipes subspe- cies and other riverine species, show that even after re- Methods ducing the target size 16 times, the alighting response Study area was about 40–55% on the small target, which is compar- The study was undertaken in the month of February able to large targets [9]. Further studies found that a 2017 on Big and Small Chamaunga Islands on Lake small target with a black cloth panel and netting mater- Victoria in western Kenya (Fig. 1). On these islands, G. f. ial each 0.25 × 0.25 m in dimensions caught more than fuscipes is the only tsetse fly species found and mainly twice the number of flies than the biconical trap, and re- inhabits the area along the lake shore [4, 9, 19, 20]. placing the black with a blue cloth panel having the Study site selection was based on the fact that G. f. fus- same dimensions slightly increased the catches [9]. The cipes populations on these islands were well documented small targets are highly efficient in reducing tsetse fly and high numbers of flies are present for meaningful ex- densities (by over 90%) and easier and cheaper to deploy perimentation [4, 21]. These islands are not inhabited by than large targets [4, 9, 11]. Apart from being exploited humans and vegetation on the lake shore consists of a for killing the tsetse fly, small targets could also be mixture of fresh water mangroves (Aeschynomene era- exploited for sampling purposes of G. f. fuscipes and phyroxylon), tropical hydrangea (Dombeya spp.) and other riverine species. tickberry (Lantana camara). Monitor lizards (Varanus Some ecological aspects critical for control of tsetse niloticus) are the main hosts for tsetse fly populations in flies, especially riverine species, include fly movement the area [4]. and spatial occupation within their restricted habitat and can only be studied with adequate sampling techniques Sampling tools [8]. Tsetse fly sampling during studies in the form of ob- Tsetse flies were caught using either biconical traps [22] servations and experiments are important for planning or sticky small targets [9, 23]. The small target com- and monitoring tsetse fly control interventions [8]. In prised of blue and black panels made from cotton cloth the last four decades, traps that attract and guide tsetse each 0.25 × 0.25 m in size and thus making it 0.25 × 0. flies into a non-return cage have been developed to serve 50 m in dimension. A board 0.25 × 0.50 m in dimension as sampling or control tools [12]. Although traps have of plywood was placed in between two small targets and biases and interpretation of catches should be done tak- fastened using staples. The board with the fastened tar- ing into account these biases; the higher proportion of gets was then covered with a transparent sticky film females to males is close to the natural sex ratio of the (Luminos 4 adhesive rolls-ungridded: Rentokil Initial tsetse fly population [8, 13, 14]. supplies, Liverpool, UK) to make sticky small targets Sticky panel traps have been mainly used to hold tsetse (Fig. 2). The sticky material on the target was not chan- flies that are killed or stunned by other sampling tools, ged over the experimental period. Both sampling tools such as electric screens [13]. They have also been used were not baited with any odour. The biconical traps had to sample G. austeni a tsetse fly species from the morsi- a radius of 0.40 m at its widest point, and a height of 1. tans group (as was the case of in the eradication of G. 30 m. austeni in Zanzibar with , SIT) [13, 15, 16]. Further, some studies have used traps and Study design, sample collection and analyses targets covered with sticky material to assess the efficacy Field experiments with the traps and sticky small targets of these as landing devices [17, 18]. were undertaken for 4 h between 08:00 and 12:00 h local The ability of a sampling tool to catch tsetse flies par- time when G. f. fuscipes are most active [3, 20], after ticularly at low density is important as it could give in- which the flies collected in the non-return cage of the formation on residual populations and lead to biconical trap and those on the sticky small target were appropriate action for planning and monitoring tsetse fly sexed and counted. On the sticky small target, flies that control interventions. A previous study showed that the landed on the blue or black panels were carefully re- small targets comprising of black and netting panels moved using forceps and placed in separate storage con- (each 0.25 × 0.25 m) caught over twice more G. f. fus- tainers colour-coded according to the part of the target cipes than the biconical trap [9]. Therefore, in this study that they landed on. The experiments were conducted we applied a sticky film to the small target having a over a period of 8 days at 4 sites. Sites 1 and 2 were on black and blue panel without any nettings, and com- Small Chamaunga and sites 3 and 4 on Big Chamaunga pared its performance as a sampling tool to the biconical (Fig. 1c). On the first and second day, experiments were Mbewe et al. Parasites & Vectors (2018) 11:268 Page 3 of 7

Fig. 1 a Study area in western Kenya. b Location of study area in Homa Bay County. c Trapping sites on Big and Small Chamaunga Islands carried out at two sites and from the third to eighth day, detransformed means) of treatments in the negative bi- the experiments were undertaken at all four sites. nomial model was obtained using the ‘effects’ package in The overall tsetse fly catches from biconical traps and R[27]. Associations were tested using Fisher’s exact test. the sticky small targets were compared using 14 repli- A Z-test was used to test for difference in proportions. cates of randomised block design experiments, each Fly catches on the sticky small targets were further ana- comprising of two adjacent days at a site as different lysed using a multiple logistic regression to measure the blocks [24]. The sampling tools as treatments were ran- association between the sex ratios of flies that landed on domly allocated to the days within the blocks and sites the blue or black panels while accounting for trap site were at least more than 100 m apart [3]. A nested survey and the day of the study. All estimates reported are ac- in the randomised block design was used to collect data companied by 95% confidence intervals and P-values less on the landing response of female and male G. f. fuscipes than 0.05 were considered statistically significant. All on the blue or black panel of the sticky small target ac- maps were created using QGIS version 2.10.1-Pisa. cording to the method developed by Vale [25]. All statistical analyses were carried out using R version Results 3.2.5 [26]. Tsetse fly catches from the sampling tools Characteristics of fly catches were modeled using a negative binomial regression to A total of 441 flies were collected, 58 (13%; 95% CI: 10– determine the catch index of the sticky small target with 16%) were caught in biconical traps while 383 (87%; 95% the biconical trap as a reference while taking into ac- CI: 84–90%) were caught by the sticky small targets. The count the block and day of study. The effect display (for overall catches for the females and males were 290 (66%; Mbewe et al. Parasites & Vectors (2018) 11:268 Page 4 of 7

Fig. 2 Sampling tools. a Sticky small target. b Biconical trap

95% CI: 61–70%) and 151 (34%; 95% CI: 30–39%), re- the sticky small target significantly caught 7.2 (95% CI: 5. spectively. The catches for the females in the biconical 3–10.1) times more flies (Z = 12.226, P < 0.0001) than the traps were 24 (41%; 95% CI: 28–54%) and those for the biconical trap after taking into account the variation due males were 34 (59%; 95% CI: 45–72%). The flies caught to the day of the experiment and the block. While accord- on the sticky small targets comprised of 266 (69%; 95% ing to sex, the sticky small target significantly caught 13.5 CI: 65–74%) females and 117 (31%; 95% CI: 26–35%) and 3.6 times more females and males, respectively com- males. Overall, there was an association between fly sex pared to the biconical trap (Z =9.551,P <0.0001andZ = and site of collection (Fisher’s exact test: P < 0.016, OR 5.978, P < 0.0001, respectively). = 0.46, 95% CI: 0.25–0.83). The total number of females caught at all four sites was higher than those of males Landing responses (Table 1). The flies caught on Small and Big Chamaunga Overall, from 383 flies that landed on the sticky small were 209 (47%; 95% CI: 42–52%) and 232 (53%; 95% CI: targets, 169 (44%; 95% CI: 39–49%) landed on the blue 48–57%) respectively and their proportions were not sig- panel while 214 (56%; 95% CI: 51–61%) landed on the nificantly different (Z-test, Z = -1.55, P = 0.121). black panel. Overall, 108 (41%; 95% CI: 35–47%) female flies landed on the blue panel and while 158 (59%; 95% Comparison of sampling tools CI: 53–65%) landed on the black panel. For male flies, Overall, the sticky small targets caught more G. f. fuscipes 61 (52%, 95% CI: 43–61%) landed on the blue panel than the biconical traps (Table 2). Detransformed means while 56 (48%, 95% CI: 39–57%) landed on the black were 3.2 (95% CI: 2.3–4.4) for the biconical trap compared panel. From all the flies that landed on the blue panel to 23.3 (95% CI: 20.6–26.4) for the sticky small target 64% (95% CI: 56–71%) were female and 36% (95% CI: (Table 2). The negative binomial regression showed that 28–43%) were male. Of the flies that landed on the black

Table 1 Fly catches at the four sites according to sex of G. f. fuscipes Island Site Female Male Total no Number % (95% CI) Number % (95% CI) Number % (95% CI) Small Chamaunga 1 97 70 (62–78) 42 30 (22–38) 139 32 (27–36) 2 36 51 (39–63) 34 49 (37–61) 70 16 (12–19) Big Chamaunga 3 55 61 (51–71) 35 39 (29–49) 90 20 (17–24) 4 102 72 (64–79) 40 28 (21–36) 142 32 (28–36) Total 290 66 (61–70) 151 34 (30–39) 441 100 Abbreviation: CI confidence interval Mbewe et al. Parasites & Vectors (2018) 11:268 Page 5 of 7

Table 2 Means and indices of catches obtained from negative binomial model for female and male G. f. fuscipes Treatment Females Males Overall Catch (95% Catch index (95% P-value Catch (95% Catch index (95% P-value Catch (95% Catch index (95% P- CI) CI) CI) CI) CI) CI) value Biconical trap 1.1 (0.6–1.9) 1 1.9 (1.2–3.1) 1 3.2 (2.3–4.4) 1 (control) Sticky small target 15.1 (12.8– 13.5 (8.2–24.4) < 6.9 (5.2–9.1) 3.6 (2.4–5.6) < 23.3 (20.6– 7.2 (5.3–10.1) < 17.8) 0.0001 0.0001 26.4) 0.0001 Abbreviation: CI confidence interval panel 73% (95% CI: 68–80%) were female while 26% biconical traps which are the most commonly used sam- (95% CI: 20–32%) were male. There was a statistically pling tools for the species. In another study, blue/black significant association between the proportion of flies targets that were 1.0 × 1.5 m in dimension covered with that landed on the blue or black panels with sex of the sticky film caught an average of about 4 to 6 times more fly (Fisher’s exact test, P < 0.044; OR: 1.59; 95% CI: 1. G. f. fuscipes than biconical traps [28]. The results from 02–2.47). A multiple logistic regression showed that fe- our study are comparable with the sticky small targets of males had a 1.71 significant increase in the likelihood of dimensions 0.25 × 0.50 m catching 7 times more flies landing on the black compared to the blue panel of the than the biconical traps suggesting that they could be an sticky small target than males and the increase could be alternative and more effective sampling tool for G. f. fus- as low as 1.07 and as high as 2.74 at 95% confidence cipes. Furthermore, the higher proportion of females interval while holding the trap site and day of study con- compared to males caught on the sticky small target is stant (Z = 2.25, P<0.025). The trap site and day of more representative of the sex ratio in the population study did not show any significant effect on the landing [13] which also makes this sampling tool more useful for response (Table 3). ecological studies. Previous versions of a sticky trap for tsetse flies were comprised of a coloured metal, wooden Discussion or cloth screen coated with sticky substance [13]. These The present study revealed that sticky small targets are have not been popular with workers due to the difficulty more efficient sampling tools for G. f. fuscipes than the in collecting and handling the flies and the poor

Table 3 Association between landing response on the blue or black panel of sticky small target with sex of G. f. fuscipes, site and day of study Explanatory variable, total (n = 383) UOR (95% CI) P-value AOR (95% CI) P-value Sex Male 1 na 1 na Female 1.59 (1.02–2.47) 0.04* 1.71 (1.07–2.74) 0.025* Site 1 1 na 1 na 2 0.47 (0.24–0.90) 0.023* 1.13 (0.36–3.58) 0.838 3 1.02 (0.57–1.83) 0.931 1.30 (0.53–3.20) 0.570 4 1.11 (0.68–1.83) 0.664 1.13 (0.63–2.01) 0.679 Day of study 1 1 na 1 na 2 0.33 (0.11–0.95) 0.040* 0.30 (0.08–1.15) 0.079 3 0.92 (0.44–1.90) 0.822 0.84 (0.37–1.92) 0.684 4 1.13 (0.48–2.66) 0.780 0.98 (0.39–2.43) 0.964 5 1.85 (0.86–4.01) 0.117 1.90 (0.82–4.41) 0.135 6 0.53 (0.20–1.44) 0.215 0.48 (0.16–1.41) 0.135 7 0.97 (0.43–2.18) 0.949 0.82 (0.34–1.98) 0.653 8 1.23 (0.51–2.95) 0.648 1 na Abbreviations: UOR unadjusted odds ratio, AOR adjusted odds ratio, CI confidence interval, na not applicable *Statistical significance at P < 0.05 Mbewe et al. Parasites & Vectors (2018) 11:268 Page 6 of 7

condition of the flies in subsequent processing such as that females show a preference to land on the black than counting and dissections [13]. As opposed to the sticky on the blue panel supports earlier practices of impreg- substances coated on the screens of the earlier sticky nating the black panel of the target with insecticide to traps, the transparent sticky film used in this study did kill females [25]. As an alternative to , bio- not present any difficulties in collecting, handling and control agents such as entomopathogenic fungi have counting the flies. Additionally, all the flies collected on been shown to be efficacious against G. f. fuscipes and the sticky small target were easily distinguished by sex, can be horizontally transmitted from infected to non- an indication that the samples were in good condition. infected tsetse flies [32, 33]. As indicated in our results, This is consistent with observations from other studies the most likely point of first contact for male flies is the that used the transparent sticky film on targets to sam- blue panel of the small target, thus it could be impreg- ple other tsetse fly species [23]. However, further studies nated with a bio-control agent. In this case, the male are needed to assess whether samples collected from flies having a preference to land on the blue than black sticky small targets could be used for dissections and panel of the small target could be contaminated and molecular biological studies. Additionally, with the serve as carriers to deliver the bio-control agent to fe- current knowledge, sticky small targets could do particu- males during . With the male fly mating more larly well in circumstances requiring highly efficient than once in its lifetime; the bio-control agent would be sampling for G. f. fuscipes such as determining its distri- transferred to each female it mates with [8]. Likewise, bution limits, mark-release-recapture experiments and though to a lesser extent, unmated females that are con- monitoring of residual populations. All these are import- taminated with the agent could also transfer it to other ant in the planning and implementation of tsetse fly males. The bio-control agent impregnated small targets control interventions. can be used in an integrated manner either concurrently Studies on the cost effectiveness of small targets indi- or sequentially with those treated with insecticide. How- cate that they use 1/24th of the material in the biconical ever, studies to determine the feasibility and cost effect- trap and are much easier to deploy [9, 29]; thus, their iveness of such integrated methods would be required. use in control of G. f. fuscipes could substantially in- Notwithstanding, exploiting landing preference of male crease field cost-effectiveness by a factor of 10 [9, 30]. In G. f. fuscipes on the blue panel in the manner suggested our study we recommend the use of the small targets could provide an opportunity for more target based inte- covered with sticky material for sampling as they signifi- grated riverine tsetse fly control strategies. cantly caught more G. f. fuscipes than biconical traps. Al- though further cost-effectiveness studies need to be Conclusion undertaken, the additional costs of the sticky material Sticky small targets significantly caught more flies than and its advantages as seen in the current study to sample biconical traps suggesting that they are more efficient fly populations and carry out ecological studies could sampling tools for G. f. fuscipes. Our study also showed justify the slight increase in costs particularly when male G. f. fuscipes preferred to first land on the blue part monitoring tsetse fly populations during control of the small target; a behaviour that could be exploited interventions. for disseminating biological control agents in popula- In order to address variations, other than those due to tions for tsetse control. the variables of interest, we used randomised block de- sign experiments, where treatments were randomly Abbreviations CI: confidence interval; HAT: human ; gHAT: Gambian assigned to experimental units in a block. This random- human African trypanosomiasis isation ensured that confounds were controlled for. Fur- ther, residual confounding was addressed during Acknowledgements statistical analysis by accounting for the block and day We thank the staff of the International Centre of Insect Physiology and Ecology for their administrative and technical support. We are also thankful the experiments were undertaken. Additionally, for as- to Mr Stanley Maramba and Mr William Owigo for their field assistance. sessment of the influence of the colour on the landing response of G. f. fuscipes according to sex, confounds Funding This study was funded by the European Union’s integrated Biological Control were addressed during analysis by accounting for the site Applied Research Programme (IBCARP) - tsetse repellent component grant and day of the study in the multiple logistic regression. number IBCARP DCI-FOOD/2014/346-739; UK’s Department for International Our results are also consistent with earlier studies Development (DFID); Swedish International Development Cooperation Agency (Sida); the Swedish Agency for Development and Cooperation (SDC); which showed that a larger proportion of G. f. fuscipes the University of Pretoria; the South African National Research Foundation’ IFRR land on the black panel of the target than the blue panel to AAY and NRF/ CRP to CWWP and the Kenyan Government. The funding [28]. Similar landing behaviour have also been reported bodies had no role in the design of the study, collection, analysis and interpretation of data and writing of the manuscript. This study is part for species belonging to the morsitans group such as G. of the postgraduate research training fellowship awarded to NJM under m. morsitans and G. pallidipes [25, 31]. The observation the Dissertation Research Internship Programme (DRIP) administered by Mbewe et al. Parasites & Vectors (2018) 11:268 Page 7 of 7

the International Centre of Insect Physiology and Ecology (icipe)and 13. Leak S, Ejigu D, Vreysen M. Collection of entomological baseline data for supported by the European Union. The views expressed herein do not tsetse area-wide integrated pest management programmes. FAO/IAEA. necessarily reflect the official opinion of the donors. Rome: FAO; 2008. 14. Challier A, Gouteux JP. Ecology and epidemiological importance of Glossina Availability of data and materials palpalis in the Ivory Coast forest zone. Insect Sci Its Appl. 1980;1:77–83. The data supporting the conclusions of this article are included within the 15. Vreysen MJB, Saleh KM, Ali MY, Abdulla AM, Zhu Z-R, Juma KG, et al. article. All datasets used and/or analysed during the current study are Glossina austeni (Diptera: Glossinidae) eradicated on the Island of Unguja, available from the corresponding author upon request. Zanzibar, using the sterile insect technique. J Econ Entomol. 2000;93:123–35. 16. Vreysen MJB, Zhu ZR, Saleh KM. Field responses of Glossina austeni to sticky Authors’ contributions panels on Unguja Island, Zanzibar. Med Vet Entomol. 1998;12:407–16. NJM, RKS, BT, JI, AAY and CWWP conceived and designed the study. NJM 17. Rayaisse JB, Kröber T, McMullin A, Solano P, Mihok S, Guerin PM. collected samples and analysed the data. NJM, RKS, BT, JI, AAY and CWWP Standardizing visual control devices for tsetse flies: West African species wrote the manuscript. All authors read and approved the final manuscript. Glossina tachinoides, G. palpalis gambiensis and G. morsitans submorsitans. PLoS Negl Trop Dis. 2012;6:e1491. Ethics approval 18. Mramba F, Oloo F, Byamungu M, Krober T, McMullin A, Mihok S, et al. Our study was conducted in conformity with the International Centre of Standardizing visual control devices for tsetse flies: East African species Insect Physiology and Ecology ethical rules for . Glossina swynnertoni. PLoS Negl Trop Dis. 2013;7(2):e2063. 19. Mwangelwa MI. 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